The levels of Mito-Vit-E in cytoplasm and mitochondria were dependant on mass spectrometry through Applied Biosystems 3200 QTRAP coupled to a Shimadzu Prominence LC (LC/MS/MS). (Mito-Vit-E), or the depletion of mitochondrial DNA abrogated VEGF-mediated mtROS creation. Overexpression of mitochondrial catalase inhibited VEGF-induced mitochondrial fat burning capacity, Rac activation, and cell migration. Furthermore, these interventions suppressed VEGF-stimulated EC migration and obstructed Rac1 activation in endothelial cells. Dynamic Rac1 reversed Mito-Vit-E-induced inhibition of EC migration Constitutively. Mito-Vit-E attenuated carotid artery reendothelialization in vivo also. These total results provide solid evidence that mtROS regulate EC migration through Rac-1. had been used. Adenovirus infections. Adenovirus that express mitochondria-targeted catalase (AdmCat) as well as the control clear adenovirus (AdNull) had been extracted from Dr. Andre Melendez through the School of Iowa Gene Transfer Vector Primary. Chlamydia of HUVEC with adenovirus was completed as previously reported on the multiplicity of infections of 100 (3, 4). Quickly, the cells had been plated and permitted to attach to the laundry overnight prior to the preferred quantity of viral contaminants was added. After 24 h, the mass media had been changed to eliminate virus as well as the cells had been cultured for another 24 h before every experiment. The performance from the infections as well as the mitochondrial deposition of mitocatalase have already been verified (3, 4). Mito-Vit-E quantification and synthesis. Mito-Vit-E was synthesized regarding to a previously released method with adjustment (39).The purity of our synthesized Mito-Vit-E was at least 90%. To verify the selective mitochondrial deposition of Mito-Vit-E, HUVEC cells at 90% confluency had been exposed to automobile or Mito-Vit-E (1 M) for 6 h before these were cleaned and gathered by trypsinization. The mitochondria and cytoplasm fractions had been made by deferential centrifugations (51). The degrees of Mito-Vit-E in cytoplasm and mitochondria had been dependant on mass spectrometry through Applied Biosystems 3200 QTRAP combined to a Shimadzu Prominence LC (LC/MS/MS). Regular curves were ready using empty cell mitochondria or homogenate spiked with various concentrations of Mito-Vit-E dissolved in DMSO. The data had been calculated towards the mitochondria/cytoplasm proportion of Mito-Vit-E focus (mass/quantity) which signifies relative selective deposition of Mito-Vit-E in mitochondria. Immunoblots. Proteins samples had been put through 4C12% gradient SDS-PAGE gels and used in polyvinylidene difluoride membranes. Membranes had been obstructed with 5% non-fat milk-Tris-buffered saline-Tween at area temperatures for 1 h and eventually probed with principal antibodies against DNA polymerase gamma (POLG; Santa Cruz Biotechnology, Santa Cruz, CA), cytochrome-oxidase (COX) II (Molecular Probes, Carlsbad, CA), phospho-Akt(Ser-473), phospho-p38MAP(Thr-180/Tyr-182), phospho- p21-turned on kinase (PAK1 Thr-423, PAK2 Thr-402), phospho-ERK1/2(T202/Y204) (Cell Signaling Technology), and GAPDH (Millipore, Temecula, CA). The membranes had been after that rinsed and incubated with matching horseradish peroxidase (HRP)-conjugated supplementary antibody (Bio-Rad, Hercules, CA). Antibody dilutions and incubation period had been regarding to manufacturer’s guidelines. Signals had been detected through the use of ChemiGlow Western world (Alpha Innotech, San Leandro, CA). POLG gene knockdown. The sequences of little interfering RNA (siRNA) are the following: POLG: 5-GGAUGGUAAUAGCUGUAAUTT-3 and 5-AUUACAGCUAUUACCAUCCTT-3; scrambled: 5-UUCUCCGAACGUGUCACGUTT-3; 5-ACGUGACACGUUCGGAGAATT-3 (Shanghai GenePharma, Shanghai, China). Transfection was performed using DharmaFECT 1 transfection reagents (Thermo Fisher Scientific, Lafayette, CO) based on the manufacturer’s guidelines. Briefly, HUVEC had been cultured in six-well plates to 80% confluence. Altogether, 200 pmol siRNA was diluted in 200 l of Opti-MEM I Decreased Serum Moderate, and 4 l of DharmaFECT 1 was diluted in 200 l from the same moderate. After 5 min, the diluted siRNA and DharmaFECT 1 had been mixed and still left at room temperatures for 20 min prior to the mix was put into the well with cells and 1.6 ml cultural moderate. After 12 h, the moderate was changed with fresh moderate with no siRNA as well as the cells had been utilized 3 or seven days following the transfection. The cells had been replated prior to the tests or if they are confluent. POLG is vital for the faithful maintenance of mtDNA (42), and POLG knockdown with siRNA continues to be used in the LDN-212854 analysis of mtDNA replication during differentiation of murine Ha sido cells (18). Cytochrome-c oxidase activity. Mitochondrial fractions had been made by differential centrifugation regarding to a previously defined method (51)..Wright GL, Maroulakou IG, Eldridge J, Liby TL, Sridharan V, Tsichlis PN, Muise-Helmericks RC. Adenovirus infections. Adenovirus that express mitochondria-targeted catalase (AdmCat) as well as the control clear adenovirus (AdNull) had LDN-212854 been extracted from Dr. Andre Melendez through the School of Iowa Gene Transfer Vector Primary. Chlamydia of HUVEC with adenovirus was completed as previously reported on the multiplicity of infections of 100 (3, 4). Quickly, the cells had been plated and permitted to attach to the laundry overnight prior to the preferred quantity of viral contaminants was added. After 24 h, the mass media had been changed to eliminate virus as well as the cells had been cultured for another 24 h before every experiment. The performance from the infections as well as the mitochondrial deposition of mitocatalase have already been verified (3, 4). Mito-Vit-E synthesis and quantification. Mito-Vit-E was synthesized regarding to a previously released method with adjustment (39).The purity of our synthesized Mito-Vit-E was at least 90%. To verify the selective mitochondrial deposition of Mito-Vit-E, HUVEC cells at 90% confluency had been exposed to automobile or Mito-Vit-E (1 M) for 6 h before these were cleaned and gathered by trypsinization. The mitochondria and cytoplasm fractions had been made by deferential centrifugations (51). The degrees of Mito-Vit-E in cytoplasm and mitochondria had been dependant on mass spectrometry through Applied Biosystems 3200 QTRAP combined to a Shimadzu Prominence LC (LC/MS/MS). Regular curves had been prepared using empty cell homogenate or mitochondria spiked with differing concentrations of Mito-Vit-E dissolved in DMSO. The info had been calculated towards the mitochondria/cytoplasm proportion of Mito-Vit-E focus (mass/quantity) which shows relative selective build up of Mito-Vit-E in mitochondria. Immunoblots. Proteins samples had been put through 4C12% gradient SDS-PAGE gels and used in polyvinylidene difluoride membranes. Membranes had been clogged with 5% non-fat milk-Tris-buffered saline-Tween at space temp for 1 h and consequently probed with major antibodies against DNA polymerase gamma (POLG; Santa Cruz Biotechnology, Santa Cruz, CA), cytochrome-oxidase (COX) II (Molecular Probes, Carlsbad, CA), phospho-Akt(Ser-473), phospho-p38MAP(Thr-180/Tyr-182), phospho- p21-triggered kinase (PAK1 Thr-423, PAK2 Thr-402), phospho-ERK1/2(T202/Y204) (Cell Signaling Technology), and GAPDH (Millipore, Temecula, CA). The membranes had been after that rinsed and incubated with related horseradish peroxidase (HRP)-conjugated supplementary antibody (Bio-Rad, Hercules, CA). Antibody dilutions and incubation period had been relating to manufacturer’s guidelines. Signals had been detected through the use of ChemiGlow Western (Alpha Innotech, San Leandro, CA). POLG gene knockdown. The sequences of little interfering RNA (siRNA) are the following: POLG: 5-GGAUGGUAAUAGCUGUAAUTT-3 and 5-AUUACAGCUAUUACCAUCCTT-3; scrambled: 5-UUCUCCGAACGUGUCACGUTT-3; 5-ACGUGACACGUUCGGAGAATT-3 (Shanghai GenePharma, Shanghai, China). Transfection was performed using DharmaFECT 1 transfection reagents (Thermo Fisher Scientific, Lafayette, CO) based on the manufacturer’s guidelines. Briefly, HUVEC had been cultured in six-well plates to 80% confluence. Altogether, 200 pmol siRNA was diluted in 200 l of Opti-MEM I Decreased Serum Moderate, and 4 l of DharmaFECT 1 was diluted in 200 l from the same moderate. After 5 min, the diluted siRNA and DharmaFECT 1 had been mixed and remaining at room temp for 20 min prior to the blend was put into the well with cells and 1.6 ml cultural moderate. After 12 h, the moderate was changed with fresh moderate with no siRNA as well as the cells had been utilized 3 or seven days following the transfection. The cells had been replated prior to the tests or if they are confluent. POLG is vital for the faithful maintenance of mtDNA (42), and POLG knockdown with siRNA continues to be used in the analysis of mtDNA replication during differentiation of murine Sera cells (18). Cytochrome-c oxidase activity. Mitochondrial fractions had been made by differential centrifugation relating to a previously referred to treatment (51). Cytochrome-oxidase activity was assessed using a industrial assay kit based on the manufacturer’s guidelines (Sigma-Aldrich, St. Louis, MO). Cytochrome-oxidase actions had been normalized by the quantity of mitochondrial proteins per response..Delivery of bioactive substances to mitochondria in vivo. mitochondrial DNA abrogated VEGF-mediated mtROS creation. Overexpression of mitochondrial catalase also inhibited VEGF-induced mitochondrial rate of metabolism, Rac activation, and cell migration. Furthermore, these interventions suppressed VEGF-stimulated EC migration and clogged Rac1 activation in endothelial cells. Constitutively energetic Rac1 reversed Mito-Vit-E-induced inhibition of EC migration. Mito-Vit-E attenuated carotid artery reendothelialization in vivo also. These results offer strong proof that mtROS regulate EC migration through Rac-1. had been used. Adenovirus disease. Adenovirus that express mitochondria-targeted catalase (AdmCat) as well as the control bare adenovirus (AdNull) had been from Dr. Andre Melendez through the College or university of Iowa Gene Transfer Vector Primary. Chlamydia of HUVEC with adenovirus was completed as previously reported in the multiplicity of disease of 100 (3, 4). Quickly, the cells had been plated and permitted to attach to the laundry overnight prior to the preferred quantity of viral contaminants was added. After 24 h, the press had been changed to eliminate virus as well as the cells had been cultured for another 24 h before every experiment. The effectiveness from the disease as well as the mitochondrial build up of mitocatalase have already been verified (3, 4). Mito-Vit-E synthesis and quantification. Mito-Vit-E was synthesized relating to a previously released method with changes (39).The purity of our synthesized Mito-Vit-E was at least 90%. To verify the selective mitochondrial build up of Mito-Vit-E, HUVEC cells at 90% confluency had been exposed to automobile or Mito-Vit-E (1 M) for 6 h before these were cleaned and gathered by trypsinization. The mitochondria and cytoplasm fractions had been made by deferential centrifugations (51). The degrees of Mito-Vit-E in cytoplasm and mitochondria had been dependant on mass spectrometry through Applied Biosystems 3200 QTRAP combined to a Shimadzu Prominence LC (LC/MS/MS). Regular curves had been prepared using empty cell homogenate or mitochondria spiked with differing concentrations of Mito-Vit-E dissolved in DMSO. The info had been calculated towards the mitochondria/cytoplasm percentage of Mito-Vit-E focus (mass/quantity) which shows relative selective build up of Mito-Vit-E in mitochondria. Immunoblots. Proteins samples had been put through 4C12% gradient SDS-PAGE gels and used in polyvinylidene difluoride membranes. Membranes had been clogged with 5% non-fat milk-Tris-buffered saline-Tween at space temp for 1 h and consequently probed with LDN-212854 major antibodies against DNA polymerase gamma (POLG; Santa Cruz Biotechnology, Santa Cruz, CA), cytochrome-oxidase (COX) II (Molecular Probes, Carlsbad, CA), phospho-Akt(Ser-473), phospho-p38MAP(Thr-180/Tyr-182), phospho- p21-triggered kinase (PAK1 Thr-423, PAK2 Thr-402), phospho-ERK1/2(T202/Y204) (Cell Signaling Technology), and GAPDH (Millipore, Temecula, CA). The membranes had been after that rinsed and incubated with related horseradish peroxidase (HRP)-conjugated supplementary antibody (Bio-Rad, Hercules, CA). Antibody dilutions and incubation period had been relating to manufacturer’s guidelines. Signals had been detected through the use of ChemiGlow Western (Alpha Innotech, San Leandro, CA). POLG gene knockdown. The sequences of little interfering RNA (siRNA) are the following: POLG: 5-GGAUGGUAAUAGCUGUAAUTT-3 and 5-AUUACAGCUAUUACCAUCCTT-3; scrambled: 5-UUCUCCGAACGUGUCACGUTT-3; 5-ACGUGACACGUUCGGAGAATT-3 (Shanghai GenePharma, Shanghai, China). Transfection was performed using DharmaFECT 1 transfection reagents (Thermo Fisher Scientific, Lafayette, CO) based on the manufacturer’s guidelines. Briefly, HUVEC had been cultured in six-well plates to 80% confluence. Altogether, 200 pmol siRNA was diluted in 200 l of Opti-MEM I Decreased Serum Moderate, and 4 l of DharmaFECT 1 was diluted in 200 l from the same moderate. After 5 min, the diluted siRNA and DharmaFECT 1 had been mixed and remaining at room temp for 20 min prior to the blend was put into the well with cells and 1.6 ml cultural moderate. After 12 h, the moderate was changed with fresh moderate with no siRNA as well as the cells had been utilized 3 or seven days following the transfection. The cells had been replated prior to the tests or if they are confluent. POLG is vital for the faithful maintenance of mtDNA (42), and POLG knockdown with siRNA continues to be used in the analysis of mtDNA replication during differentiation of murine Ha sido cells (18). Cytochrome-c oxidase activity. Mitochondrial fractions had been made by differential centrifugation regarding to a previously defined method (51). Cytochrome-oxidase activity was assessed using a industrial assay kit based on the manufacturer’s guidelines (Sigma-Aldrich, St. Louis, MO). Cytochrome-oxidase actions had been normalized by the quantity of mitochondrial proteins per reaction. Recognition of mitochondrial ROS. Mitochondrial superoxide was discovered by labeling the cells with MitoSox Crimson (Invitrogen, Carlsbad, CA). HUVEC had been packed with MitoSox Crimson (28) (5 M, 20 min) and activated with VEGF (50 ng/ml, 5 min) or automobile before stream cytometry. To identify mitochondrial H2O2, HUVEC had been transfected using a plasmid transiently, pHyPer-dMito, which encodes a derivative of hydrogen peroxide-specific.Mito-Vit-E also attenuated carotid artery reendothelialization in vivo. or the depletion of mitochondrial DNA abrogated VEGF-mediated mtROS creation. Overexpression of mitochondrial catalase also inhibited VEGF-induced mitochondrial fat burning capacity, Rac activation, and cell migration. Furthermore, these interventions suppressed VEGF-stimulated EC migration and obstructed Rac1 activation in endothelial cells. Constitutively energetic Rac1 reversed Mito-Vit-E-induced inhibition of EC migration. Mito-Vit-E also attenuated carotid artery reendothelialization in vivo. These outcomes provide strong proof that mtROS regulate EC migration through Rac-1. had been used. Adenovirus an infection. Adenovirus that express mitochondria-targeted catalase (AdmCat) as well as the control unfilled adenovirus (AdNull) had been extracted from Dr. Andre Melendez through the School of Iowa Gene Transfer Vector Primary. Chlamydia of HUVEC with adenovirus was completed as previously reported on the multiplicity of an infection of 100 (3, 4). Quickly, the cells had been plated and permitted to attach to the laundry overnight prior to the preferred quantity of viral contaminants was added. After 24 h, the mass media had been changed to eliminate virus as well as the cells had been cultured for another 24 h before every experiment. The performance from the an infection as well as the mitochondrial deposition of mitocatalase have already been verified (3, 4). Mito-Vit-E synthesis and quantification. Mito-Vit-E was synthesized regarding to a previously released method with adjustment (39).The purity of our synthesized Mito-Vit-E was at least 90%. To verify the selective mitochondrial deposition of Mito-Vit-E, HUVEC cells at 90% confluency had been exposed to automobile or Mito-Vit-E (1 M) for 6 h before these were cleaned and gathered by trypsinization. The mitochondria and cytoplasm fractions had been made by deferential centrifugations (51). The degrees of Mito-Vit-E in cytoplasm and mitochondria had been dependant on mass spectrometry through Applied Biosystems 3200 QTRAP combined to a Shimadzu Prominence LC (LC/MS/MS). Regular curves had been prepared using empty cell homogenate or mitochondria spiked with differing concentrations of Mito-Vit-E dissolved in DMSO. The info had been calculated towards the mitochondria/cytoplasm proportion of Mito-Vit-E focus (mass/quantity) which signifies relative selective deposition of Mito-Vit-E in mitochondria. Immunoblots. Proteins samples had been put through 4C12% gradient SDS-PAGE gels and used in polyvinylidene difluoride membranes. Membranes had been obstructed with 5% non-fat milk-Tris-buffered saline-Tween at area heat range for 1 h and eventually probed with principal antibodies against DNA polymerase gamma (POLG; Santa Cruz Biotechnology, Santa Cruz, CA), cytochrome-oxidase (COX) II (Molecular Probes, Carlsbad, CA), phospho-Akt(Ser-473), phospho-p38MAP(Thr-180/Tyr-182), phospho- p21-turned on kinase (PAK1 Thr-423, PAK2 Thr-402), phospho-ERK1/2(T202/Y204) (Cell Signaling Technology), and GAPDH (Millipore, Temecula, CA). The membranes had been after that rinsed and incubated with matching horseradish peroxidase (HRP)-conjugated supplementary antibody (Bio-Rad, Hercules, CA). Antibody dilutions and incubation period had been regarding to manufacturer’s guidelines. Signals had been detected through the use of ChemiGlow Western world (Alpha Innotech, San Leandro, CA). POLG gene knockdown. The sequences of little interfering RNA (siRNA) are the following: POLG: 5-GGAUGGUAAUAGCUGUAAUTT-3 and 5-AUUACAGCUAUUACCAUCCTT-3; scrambled: 5-UUCUCCGAACGUGUCACGUTT-3; 5-ACGUGACACGUUCGGAGAATT-3 (Shanghai GenePharma, Shanghai, China). Transfection was performed using DharmaFECT 1 transfection reagents (Thermo Fisher Scientific, Lafayette, CO) based on the manufacturer’s guidelines. Briefly, HUVEC had been cultured in six-well plates to 80% confluence. Altogether, 200 pmol siRNA was diluted in 200 l of Opti-MEM I Decreased Serum Moderate, and LDN-212854 4 l of DharmaFECT 1 was diluted in 200 l from the same moderate. After 5 min, the diluted siRNA and DharmaFECT 1 had been mixed and still left at room heat range for 20 min prior to the mix was put into the well with cells and 1.6 ml cultural moderate. After 12 h, the moderate was changed with fresh moderate with no siRNA as well as the cells had been used 3 or 7 days after the transfection. The cells were replated before the experiments or when they are confluent. POLG is essential for the faithful maintenance of mtDNA (42), and POLG knockdown with siRNA has been used in the study of mtDNA replication during differentiation of murine ES cells (18). Cytochrome-c oxidase activity. Mitochondrial fractions were prepared by differential centrifugation according to a previously explained process (51). Cytochrome-oxidase activity was measured using a commercial assay kit according to the manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO). Cytochrome-oxidase activities were normalized by the amount of mitochondrial protein per reaction. Detection of mitochondrial ROS. Mitochondrial superoxide was detected by labeling the cells with MitoSox Red (Invitrogen, Carlsbad, CA). HUVEC were loaded with MitoSox Red (28) (5 M, 20 min) and stimulated with VEGF (50 ng/ml, 5 min) or vehicle before circulation cytometry. To detect mitochondrial H2O2, HUVEC were transiently transfected with a plasmid, pHyPer-dMito, which encodes a derivative of hydrogen peroxide-specific sensor protein Hyper (9) tagged with mitochondrial transmission peptide sequence (Evrogen, Moscow, Russia). Cells were produced in fibronectin-coated glass-bottom microwell dishes (Ashland, MA) and the transfection was done with FuGENE 6 (Roche Applied Science). Cells were serum starved overnight before mitochondrial H2O2.In addition to that, Mito-Vit-E and mtDNA depletion inhibited VEGF-induced mitochondrial and intracellular ROS production. of the antioxidant, vitamin E (Mito-Vit-E), or the depletion of mitochondrial DNA abrogated VEGF-mediated mtROS production. Overexpression of mitochondrial catalase also inhibited VEGF-induced Rabbit Polyclonal to ERAS mitochondrial metabolism, Rac activation, and cell migration. Furthermore, these interventions suppressed VEGF-stimulated EC migration and blocked Rac1 activation in endothelial cells. Constitutively active Rac1 reversed Mito-Vit-E-induced inhibition of EC migration. Mito-Vit-E also attenuated carotid artery reendothelialization in vivo. These results provide strong evidence that mtROS regulate EC migration through Rac-1. were used. Adenovirus contamination. Adenovirus that express mitochondria-targeted catalase (AdmCat) and the control vacant adenovirus (AdNull) were obtained from Dr. Andre Melendez through the University or college of Iowa Gene Transfer Vector Core. The infection of HUVEC with adenovirus was carried out as previously reported at the multiplicity of contamination of 100 (3, 4). Briefly, the cells were plated and allowed to attach to the dishes overnight before the desired amount of viral particles was added. After 24 h, the media were changed to remove virus and the cells were cultured for another 24 h before each experiment. The efficiency of the contamination and the mitochondrial accumulation of mitocatalase have been confirmed (3, 4). Mito-Vit-E synthesis and quantification. Mito-Vit-E was synthesized according to a previously published method with modification (39).The purity of our synthesized Mito-Vit-E was at least 90%. To confirm the selective mitochondrial accumulation of Mito-Vit-E, HUVEC cells at 90% confluency were exposed to vehicle or Mito-Vit-E (1 M) for 6 h before they were washed and harvested by trypsinization. The mitochondria and cytoplasm fractions were prepared by deferential centrifugations (51). The levels of Mito-Vit-E in cytoplasm and mitochondria were determined by mass spectrometry by the use of Applied Biosystems 3200 QTRAP coupled to a Shimadzu Prominence LC (LC/MS/MS). Standard curves were prepared using blank cell homogenate or mitochondria spiked with varying concentrations of Mito-Vit-E dissolved in DMSO. The data were calculated to the mitochondria/cytoplasm ratio of Mito-Vit-E concentration (mass/volume) which indicates relative selective accumulation of Mito-Vit-E in mitochondria. Immunoblots. Protein samples were subjected to 4C12% gradient SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% nonfat milk-Tris-buffered saline-Tween at room temperature for 1 h and subsequently probed with primary antibodies against DNA polymerase gamma (POLG; Santa Cruz Biotechnology, Santa Cruz, CA), cytochrome-oxidase (COX) II (Molecular Probes, Carlsbad, CA), phospho-Akt(Ser-473), phospho-p38MAP(Thr-180/Tyr-182), phospho- p21-activated kinase (PAK1 Thr-423, PAK2 Thr-402), phospho-ERK1/2(T202/Y204) (Cell Signaling Technology), and GAPDH (Millipore, Temecula, CA). The membranes were then rinsed and incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibody (Bio-Rad, Hercules, CA). Antibody dilutions and incubation time were according to manufacturer’s instructions. Signals were detected by using ChemiGlow West (Alpha Innotech, San Leandro, CA). POLG gene knockdown. The sequences of small interfering RNA (siRNA) are as follows: POLG: 5-GGAUGGUAAUAGCUGUAAUTT-3 and 5-AUUACAGCUAUUACCAUCCTT-3; scrambled: 5-UUCUCCGAACGUGUCACGUTT-3; 5-ACGUGACACGUUCGGAGAATT-3 (Shanghai GenePharma, Shanghai, China). Transfection was performed using DharmaFECT 1 transfection reagents (Thermo Fisher Scientific, Lafayette, CO) according to the manufacturer’s instructions. Briefly, HUVEC were cultured in six-well plates to 80% confluence. In total, 200 pmol siRNA was diluted in 200 l of Opti-MEM I Reduced Serum Medium, and 4 l of DharmaFECT 1 was diluted in 200 l of the same medium. After 5 min, the diluted siRNA and DharmaFECT 1 were mixed and left at room temperature for 20 min before the mixture was added to the well with cells and 1.6 ml cultural medium. After 12 h, the medium was replaced with fresh medium without the siRNA and the cells were used 3 or 7 days after the transfection. The cells were replated before the experiments or when they are confluent. POLG is essential for the faithful maintenance of mtDNA (42), and POLG knockdown with siRNA has been used in the study of mtDNA replication during differentiation of murine ES cells (18). Cytochrome-c oxidase activity. Mitochondrial fractions were prepared by differential centrifugation according to a previously described procedure (51). Cytochrome-oxidase activity was measured using a commercial assay kit according to the manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO). Cytochrome-oxidase activities were normalized by the amount of mitochondrial protein per reaction. Detection of mitochondrial ROS. Mitochondrial superoxide was detected by labeling the cells with MitoSox Red (Invitrogen, Carlsbad, CA). HUVEC were loaded with MitoSox Red (28) (5 M, 20 min) and stimulated with VEGF (50 ng/ml, 5 min) or vehicle before flow cytometry. To detect mitochondrial H2O2, HUVEC were transiently transfected with a plasmid, pHyPer-dMito, which encodes a.